A founder mutation causing a severe ... - CENTOGENE

Brief Communication
A founder mutation causing a severe methylenetetrahydrofolate reductase (MTHFR)
deficiency in Bukharian Jews
Shay Ben-Shachar a , Tal Zvi a , Arndt Rolfs b,c , Andrea Breda Klobus c , Yuval Yaron a,d ,
Anat Bar-Shira a , Avi Orr-Urtreger a,d, ⁎
a The Genetic Institute & Prenatal Diagnosis Unit, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
b Albrecht-Kossel-Institute for Neuroregeneration, Medical Faculty, University of Rostock, Germany
c Centogene GmbH, The Rare Disease Company, Rostock, Germany
d Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
article info
Article history:
Received 30 June 2012
Received in revised form 9 August 2012
Accepted 9 August 2012
Available online 18 August 2012
Keywords:
MTHFR
Founder mutation
Bukhara
1. Introduction
abstract
Homocystinuria due to methylenetetrahydrofolate reductase
(MTHFR) deficiency (OMIM ID: 236250) is a rare inborn error of folate
metabolism resulting in elevated homocysteine levels in plasma and
urine [1]. Methylenetetrahydrofolate reductase catalyzes the conversion
of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate
for homocysteine remethylation to methionine. The severe
form of the disease is characterized by developmental delay, seizures and
microcephaly [2], and may be associated with infantile epilepsy [3].The
disease is autosomally recessively inherited, caused by mutations in the
MTHFR gene [4]. It has been suggested that early treatment with betaine
may alleviate the effect of the disease to the brain in some cases [5].
In this study we demonstrate the existence of a common splicing
founder mutation in the MTHFR gene, causing a severe MTHFR deficiency
among individuals of Jewish Bukharic ancestry.
2. Material and methods
2.1. Patient recruitment
The study protocol and informed consent were approved by the
local Institutional Ethical Committee. DNA and RNA samples were
collected from affected individuals and their parents.
⁎ Corresponding author at: The Genetic Institute, Tel Aviv Sourasky Medical Center,
6 Weizmann St., Tel Aviv, 64239, Israel. Fax: +972 3 6974555.
E-mail address: aviorr@tasmc.health.gov.il (A. Orr-Urtreger).
1096-7192/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ymgme.2012.08.011
Molecular Genetics and Metabolism 107 (2012) 608–610
Contents lists available at SciVerse ScienceDirect
Molecular Genetics and Metabolism
journal homepage: www.elsevier.com/locate/ymgme
Methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare autosomal recessive disorder. A novel
homozygous MTHFR c.474A>T (p.G158G) mutation was detected in two unrelated children of Jewish
Bukharian origin. This mutation generates an abnormal splicing and early termination codon. A carrier
frequency of 1:39 (5/196) was determined among unrelated healthy Bukharian Jews. Given the disease
severity and allele frequency, a population screening for individuals of this ancestry is warranted in order to
allow prenatal, or preimplantation diagnosis.
© 2012 Elsevier Inc. All rights reserved.
2.2. MTHFR gene sequencing
MTHFR gene (NM_005957.4) was screened for mutations using
standard PCR and sequencing of both DNA strands of the coding
region and the exon–intron boundaries followed the CMGS (Clinical
Molecular Genetics Society) best practice guidelines (Centogene
GmbH, Rostock, Germany). The mutation has been deposited in the
NCBI dbSNP database (rs199476142).
2.3. RNA analysis
RT-PCR (reverse transcriptase PCR) was performed on cDNA
generated from RNA extracted from peripheral blood leukocytes
of the patients. Primers for the RT-PCR were located in exon 2
and 4 of the reference sequence to verify whether exon 3 and/or
intron 3 are included in the mutated transcript of the patient
and the carrier parents. The primers' sequences are available upon
request.
2.4. Determination of the c.474A>T frequency among controls
We examined 196 unaffected and unrelated Bukharian Jews
and 176 Ashkenazi Jews, by an allelic discrimination test (TaqMan,
custom-made test, Applied Biosystems, Carlsbad, CA, USA). The
carrier status was further confirmed in all these individuals by Sanger
sequencing.

3. Results
A 13-month-old boy was referred for genetic work-up with the
diagnosis of MTHFR deficiency. He had microcephaly, seizures and
severe developmental delay. MTHFR deficiency was suspected due to
elevated homocysteine in the blood and urine, and was confirmed by
testing the specific activity of methylenetetrahydrofolate reductase
in extract of the patient's fibroblasts (0.05 nmol/mg protein/h). The
parents were non-consanguineous Jews of Bukharian ancestry. They
previously had a daughter with similar manifestations, who died
at 7 months of age. The analyses revealed a previously unreported
homozygous variant c.474A>T (p.G158G) that caused a synonymous
change. The parents were found to be heterozygous carriers of this
change (Figs. 1A–C).
The same homozygous mutation was detected in a 16-month-old
girl, referred to our molecular genetics laboratory, with similar neurological
and biochemical findings, suggestive of MTHFR deficiency.
This girl was also an offspring of a non-consanguineous couple of
Bukharian Jewish ancestry, unrelated to the family previously described.
Her parents were found to be heterozygous carriers of the same
mutation.
As the transversion is located at the 3′ extremity of exon 3, and
does not alter an amino acid, aberrant splicing was considered to be a
consequence of this alteration.
To test the hypothesis that the c.474A>T mutation causes an
aberrant splicing, we performed an expression study by RT-PCR. From
the cDNA of the affected individuals we found an amplicon of about
1350 base pairs (bp), which was approximately 850 bp longer than
the ~500 bp amplicon obtained in negative controls (Fig. 1F). The
heterozygous parents had signs of both the wild-type allele and the
mutant allele. Sanger sequencing of the large amplicon revealed
the inclusion of the entire 838 bp sized intron 3 of the MTHFR gene
A
B
C
Genomic DNA
Exon 3
S. Ben-Shachar et al. / Molecular Genetics and Metabolism 107 (2012) 608–610
Intron 3
(Figs. 1D and E). An early stop codon was generated in the novel
aberrant transcript 22 codons downstream to the original exon–
intron boundary, confirming the truncation nature of the mutation.
As these two affected individuals belong to the isolated ethnic group
of the Jews coming from the province of Bukhara (Uzbekistan), we
hypothesized that the c.474A>T mutation could be a founder allele. We
therefore examined additional 196 unaffected and unrelated Bukharian
Jews for the c.474A>T mutation and found it in five individuals,
providing an estimated carrier frequency of 1:39. The mutation was
not found among 176 Ashkenazi Jews. The prevalence of MTHFR
deficiency due to this founder mutation in Bukharian Jews is expected
to be ~1:6000.
4. Discussion
Our results show that a previously unreported mutation c.474A>T
(p. G158G) is a common founder mutation among Jews of Bukharian
ancestry, leading to aberrant splicing and resulting in generation of a
premature termination codon. Currently, population-based screening
programs are offered for a number of common genetic disorders
world-wide, and are particularly prevalent in Israel [6]. Several
criteria have been proposed to select genetic disorders for population
screening. The agreed criteria include severity of disease, early onset,
and high frequency of carriers, availability of genetic counseling and
prenatal diagnosis, and an existence of a reliable test [7]. The MTHFR
deficiency due to the c.474A>T mutation among couples of Bukharian
Jewish origin clearly fulfills these criteria. Therefore, a populationbased
screening for the disease among this population is warranted,
and will be added to the panel of ethnicity-specific mutations that
are being offered in Israel. Furthermore, our results emphasize the
importance of identifying rare founder mutations that lead to severe
diseases among genetically-isolated populations. Such an approach
Wild type
Affected
Carrier
D
E
F
1517bp
1200bp
1000bp
500bp
cDNA
Exon 3 Exon 4
Exon 3
Intron 3
Fig. 1. Molecular analysis of the c.474A>T (G158G) mutation. On genomic DNA, compared with controls (A), affected individuals have a homozygous A>T transversion (B), while
carrier individuals show the same change in the heterozygous state (C). The exon–intron junction is defined by the black vertical line. At the cDNA level, compared with unaffected
controls (D), the original intron 3 is included in the cDNA of affected individuals immediately after exon 3 (E). RT-PCR of a cDNA fragment including exons 3 and 4 of the reference
sequence revealed an expected ~500 bp band in controls and an enlarged ~1350 bp band in affected individuals. Both bands were detected in the heterozygous parents (F).
609

610 S. Ben-Shachar et al. / Molecular Genetics and Metabolism 107 (2012) 608–610
will assist in preventing and alleviating the burden of severe rare
genetic disorders, in addition to the prevention of common severe
recessive diseases, such as spinal muscular atrophy (SMA) [8].
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Web-based resources
http://www.ncbi.nlm.nih.gov/omim
http://www.ncbi.nlm.nih.gov/nuccore
http://www.ncbi.nlm.nih.gov/projects/SNP/
http://www.ensembl.org
http://www.ncbi.nlm.nih.gov/projects/SNP/tranSNP/vsub.cgi?rm=view&set_id=23454